Ageing Increases Cardiac Electrical Remodelling in Rats and Mice via NOX4/ROS/CaMKII-Mediated Calcium Signalling

Objective Ageing is one of the risk factors associated with cardiovascular diseases including cardiac arrhythmias and heart failure. Ageing-related cardiac dysfunction involves a complicated pathophysiological progress. Abnormal membrane voltage and Ca2+ dynamics in aged cardiomyocytes contribute to ageing-related arrhythmias. However, its underlying mechanisms have not been well clarified. Methods Young and old rats or mice were included in this study. Cardiac electrophysiological properties and functions were assessed by ECG, echocardiography, and ex vivo heart voltage and Ca2+ optical mapping. Proteomics, phosphor-proteomics, Western blotting, Masson staining, and ROS measurement were used to investigate the underlying mechanisms. Results Ageing increased the incidence of cardiac hypertrophy and fibrosis in rats. Moreover, ageing increased the occurrence of ventricular tachycardia or ventricular fibrillation induced by rapid pacing and during isoprenaline (ISO) (1 mg/kg i.p.) challenge in mice in vivo. Optical mapping with dual dyes (membrane voltage (Vm) dye and intracellular Ca2+ dye) simultaneously recording revealed that ageing increased the action potential duration (APD) and Ca2+ transient duration (CaTD) and slowed the ventricular conduction with the Langendorff-perfused mouse heart. More importantly, ageing increased the ISO-induced (1 μM) changes of APD (ΔAPD80) and CaTD (ΔCaTD50). Ageing also delayed the decay of Ca2+ transient by extending the decay time constant from 30% to 90% (τ30−90). In addition, ageing decreased the Vm/Ca2+ latency which represented the coupling of Vm/Ca2+ including between the midpoint of AP depolarization and Ca2+ upstroke, peak transmembrane voltage and peak cytosolic calcium, and time to 50% voltage repolarization and extrusion of cytosolic calcium. Optical mapping also revealed that ageing increased the ISO-induced arrhythmia incidence and occurrence of the excitation rotor. Proteomics and phosphor-proteomics assays from rat hearts demonstrated ageing-induced protein and phosphor-protein changes, suggesting that CaMKII was involved in ageing-induced change. Ageing increased the level of ROS and the expression of NOX4, oxidative CaMKII (ox-CaMKII), phosphorated CaMKII (p-CaMKII), and periostin. Conclusion Ageing accelerates cardiac remodelling and increases the susceptibility to ventricular arrhythmias through NOX4/ROS/CaMKII pathway-mediated abnormal membrane voltage and intracellular Ca2+ handling and Vm/Ca2+ coupling.


Introduction
Ageing has been demonstrated to be one of the major risk factors for cardiovascular disease (CVD) [1,2], including the incidence of hypertension, arrhythmias, and heart failure, which contribute to higher morbidity and mortality in the elder population. Within the elderly people (>70 years), the proportion of total deaths worldwide due to CVD will reach up to 40% in 2030 from the World Health Organization (WHO) [3]. The heart has complicated pathophysiological changes during ageing. Considerable evidence implicates that endothelial dysfunction, mitochondrial oxidative stress, chromatin remodelling, and genomic instability are involved in vascular and cardiac dysfunction in elderly patients [4,5].
Ageing is associated with the increased predisposition of arrhythmias through complicated structural and functional alterations in cardiac mechanical and electrical systems, as well as energetics and metabolism [1]. Myocardium hypertrophy and interstitial fibrosis-resulting in altered cellular coupling and exaggerated directional differences in conduction (anisotropy)-increase heterogeneity in impulse propagation properties and refractoriness of the myocardium [6,7].
Mitochondria are the critical organelle for the generation of metabolic energy in eukaryotic cells through oxidative phosphorylation. More than 90% of the intracellular ATP consumed by cardiomyocytes was generated in mitochondria. The dysfunction of mitochondria has been demonstrated to be involved in multiple heart diseases [8][9][10][11]. The increase in oxidative stress due to the increase in reactive oxygen species (ROS) production with age results in an overall enhancement in the rate of cardiomyocyte death and increased fibrosis. Oxidative stress is believed to be an independent mediator of age-related arrhythmias through reducing the repolarization reserve and formation of early and delay afterdepolarizations due to activation of the Na + -Ca 2+ exchanger. Lots of studies also revealed that Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) activation contributed to a variety of cardiac diseases including heart failure and arrhythmias [12]. ROS and CaMKII played a crucial role in the development of arrhythmias. Xie et al. found that ROS-induced arrhythmias were blocked through inhibiting CaMKII [13]. It has been shown that the increased oxidative stress prolonged diastolic relaxation through oxidative damage of the sarcoplasmic reticulum SERCA ATP pump and decreased its Ca 2+ -sequestering activity [14]. The normal coupling and dynamics of membrane voltage (V m ) and intracellular Ca 2+ contributed to integrated cardiac electrophysiological function, whereas its abnormal coupling was involved in the occurrence of arrhythmias [15]. However, the molecular bases and underlying mechanisms for the increased susceptibility to arrhythmias in the elderly are not fully understood and need further exploration.
In this present study, we explored the role of oxidative stress in increased susceptibility to ventricular arrhythmias in ageing hearts and its underlying signalling pathway. We revealed the abnormal coupling of V m and intracellular Ca 2+ handing in ageing animals under ISO-induced β adrenergic stress conditions.

Methods and Materials
2.1. Animals. Young (4 months) and old (

Echocardiography and Hemodynamic Analysis in Intact
Hearts. WKY rats were terminally anaesthetized with 2% isoflurane using a gas anaesthesia machine (RWD Life Science, Shenzhen city, Guangdong province, China). Parasternal short-axis section (PSAX) M-mode echocardiography was recorded using the Vevo®3100 micro-ultrasound imaging system (FUJIFILM VisualSonics Inc., Canada) following the manufacturer's instructions. Three measurements were taken at end-systole (s) and end-diastole (d) which were averaged to calculate the corresponding values of the intraventricular septal thickness (IVSs and IVSd), left ventricular posterior wall thickness (LVPWs and LVPWd), and left ventricular end-diastolic (LVIDd) and end-systolic dimensions (LVIDs). The ejection fraction (EF) and fractional shortening (FS) were also acquired from the recorded measurements.

Masson's Trichrome Staining.
Hearts were removed from WKY rats anaesthetized with 2-5% isoflurane and transferred to 4% polyformaldehyde in PBS for fixation. 5 μm paraffin sections were stained using Masson's trichrome method as described [16]. Images were acquired using PANNORAMIC SCAN (3DHISTECH Ltd., Hungary) to scan the whole film. The mean cross-sectional area of cardiomyocytes was calculated through approximately 400 randomly selected cells using CaseViewer software (version 2.3, 3DHISTECH Ltd., Hungary). The collagen volume fraction was measured through 20 images from different fields by ImageJ software (version 1.53e).

Electrocardiography (ECG) and Programmed Electrical
Stimulation (PES). Surface ECG recording of C57/BL mice in vivo was monitored using a multichannel physiological instrument (MP150, BIOPAC Systems Inc., USA) under anaesthesia with 0.5-1% isoflurane using the gas anaesthesia machine. To assess propensity to ventricular arrhythmias, anaesthetized mice were subjected to PES in vivo. Briefly, the heart was exposed and a stimulus electrode was placed on the surface of the heart and connected to the stimulator (SEN-7203, Nihon Kohden, Japan). Mice were subjected to burst pacing for 50 beats with cycle lengths (CLs) of 90, 70, 50, and 30 ms to induce the arrhythmia. Ventricular tachycardia with regular waveforms was defined as VT, while VF was characterized by irregular fibrillating waveforms. Intraperitoneal injection of 1 mg/kg isoproterenol (ISO) (cat: HY-B0448, MedChemExpress, USA) was performed to increase the susceptibility to arrhythmia.

Optical
Mapping in Intact Hearts. The electrophysiological function and arrhythmia susceptibility of intact heart ex vivo were assessed using the optical mapping system equipped with an EMCCD camera according to our previous research [17,18]. In brief, C57/BL mice were anaesthetized with 2-5% isoflurane followed by heparin injection (200 units intraperitoneal injection). The heart was removed and placed in cold PSS equilibrated with 95% O 2 and 5% CO 2 . Using the Langendorff equipment, the aorta perfusion

Ageing Increased Cardiac Hypertrophy and Fibrosis.
We first explored the cardiac functions and basic characters of hearts from young and old WKY rats by echocardiography and Masson staining as shown in Figure 1. Echocardiography and hemodynamic analysis showed that IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs were increased in old rats (Figures 1(a) and 1(b)). IVSd, IVSs, LVIDs, and LVPWd have a statistical difference between the two groups (P = 0:017, P = 0:015, P = 0:031, and P = 0:032, respectively), while EF and FS in old rats were slightly decreased with no statistical difference (P = 0:103, P = 0:051). In addition, we found heart enlargement and collagen deposition in histology. Because the older mice gained weight, there was no significant increase in the HW/BW ratio between young and old rats (P = 0:070). However, the cross-sectional area and collagen volume were increased in the old group (both P ≤ 0:001). Furthermore, the expression of periostin, which is one of the markers of cardiac fibrosis, also significantly increased in the old group (P ≤ 0:001, Figures 1(g) and 1 (h)). These data suggest that ageing exacerbated cardiac hypertrophy and fibrosis. These data suggest that ageing exacerbated cardiac hypertrophy and fibrosis.

Ageing Increased the Susceptibility to Ventricular
Arrhythmias Induced by Rapid Pacing In Vivo. Previous studies have demonstrated that cardiac fibrosis exists in various cardiovascular diseases and accelerates the occurrence and maintenance of cardiac arrhythmias [15,20]. Therefore, we further investigated the effects of ageing on the susceptibility to ventricular arrhythmias using surface ECG and PES with a cycle length from 90 ms to 30 ms to induce VT in C57/BL mice in vivo. At baseline, there was no significant difference in ECG basic characteristics between the young and old groups ( Figure 2). There was also no significant difference in the incidence of arrhythmia with PES between the young (mm)  Oxidative Medicine and Cellular Longevity (10.81%, n = 4/37) and old (16.22%, n = 6/37) groups in the baseline (P = 0:496). However, after acute intraperitoneal injection of ISO (1 mg/kg) for 5 min, the incidence of VT in the old group (51.35%, n = 19/37) was significantly increased than that in the young group (21.62%, n = 8/37) (P = 0:008).
The data suggest that the old mice are more susceptible to cardiac arrhythmia under the acute ISO-induced β adrenergic stress condition.

Ageing Induced Abnormal Action Potential (AP) and
Ca 2+ Signal. Cardiac fibrosis can cause abnormal electrical activity and conduction, which contributed to cardiac arrhythmia [6,21,22]. Therefore, we explored optical mapping to investigate the effects of ageing on AP and the Ca 2+ signal and their coupling with programmed electrical stimulation (PES) with PCL of 100 ms (pacing frequency: 10 Hz) at the cardiac apex as shown in Figures 3 and 4. The APD80 (the duration from the peak of AP to repolarization at 80%) (Figure 3(a)) and CaTD50 (the duration from the time of the maximum upstroke velocity to decay at 50% of Ca 2+ transient) (Figure 3(g)) were quantified as AP duration and Ca 2+ transient duration, respectively. We found that the APD80 in old mice was significantly prolonged from 36:31 ± 2:28 ms to 51:10 ± 3:81 ms (P ≤ 0:001) pacing at 10 Hz (Figure 3(c)). After ISO perfusion, the APD80 was shortened in both young and old mice (27:94 ± 1:94 ms in the young group, 38:63 ± 1:78 ms in the old group, P = 0:010) and it is interesting that the ISO-induced decrease of APD80 (quantified as ΔAPD80) was more obvious in the old mice with ΔAPD80 from −8.38 ± 0.93 ms to −12.46 ± 3.03 ms (Figure 3(d)).
We further analyzed the calculation of "conduction velocity" (CV) to provide a measure of AP activation spread and conduction across the whole tissue according to the previous protocol [19] as shown in Figure 3(e). We found that CV was decreased from 77:46 ± 1:71 cm/s in young mice to 64:93 ± 3:12 cm/s in old mice pacing at 10 Hz (P = 0:042), Figures 3(e) and 3(f)). These data suggest that ageing inhibits the AP spread and conduction.
Abnormal coupling of membrane voltage (V m ) and intracellular Ca 2+ transient dynamics is the important mechanism of cardiac arrhythmias [15]. To explore Ca 2+ and V m clock coupling and how ageing may affect such coupling, we    7 Oxidative Medicine and Cellular Longevity analyzed the voltage-calcium latency as optical mapping was simultaneously recorded in V m and Ca 2+ at upstroke, peak, and decay time points as shown in Figures 4(a)-4(d). Old mice demonstrated a significantly shortened latency time between the midpoint of AP depolarization and Ca 2+ upstroke (6:81 ± 0:27 ms in the young group, 5:91 ± 0:29 m s in the old group, P = 0:029), peak transmembrane voltage and peak cytosolic calcium (9:67 ± 0:30 ms in young group, 7:87 ± 1:31 ms in the old group, P = 0:010), and time to 50% voltage repolarization and extrusion of cytosolic calcium (13:44 ± 1:15 ms in the young group, 2:33 ± 0:63 ms in the old group, P ≤ 0:001).
In addition, we explored the arrhythmia incidence with optical mapping following 1 μM ISO treatment pacing at 50 Hz ex vivo. Old mice presented a high incidence of VT events (Figure 4(e)). Under baseline without treatment of ISO, both young and old mice did not have arrhythmias. However, 1 μM ISO induced 100% (5/5) VT occurrence in old mice, while there was no arrhythmia in young mice treated with ISO (Figure 4(f)). These data were consistent with the surface ECG recording in vivo (Figure 2). The heat map showed the detail of the rotor and the process of VT (Figure 4(g)).
All the data with optical mapping suggest that ageing increases the arrhythmia incidence through increasing the AP and Ca 2+ duration and shortening the latency.

The Effects of Ageing on Proteomics and
Phosphoproteomics. To further identify the specific mechanism of age-induced fibrosis leading to arrhythmias, we used The typical CaTD 50 map and calcium transient trace (young: black line; old: red line) in intact Langendorff perfused young and old mouse hearts before and following 1 μM ISO challenge in pacing at 10 Hz. (i) Quantitative analysis of CaTD 50 from individual hearts in young and old mice before and following 1 μM ISO * P < 0:05, * * P < 0:01, and * * * P < 0:001). (j) Ageing induced fractional changes in APD 80 challenged by ISO (ΔCaTD 50 ) (P > 0:05). (k) The representative CaTD map shows the calculation of decay constant (τ) by fitting of exponential decay points between 30% and 90% decay from the peak (τ 30-90 ), illustrated for the regional signal in red. (l) Histogram showing the τ 30-90 in young and old mice before and following 1 μM ISO pacing at 10 Hz ( * P < 0:05 and * * P < 0:01). n = 5 (each group). 8 Oxidative Medicine and Cellular Longevity proteomics and phosphoproteomics to analyze changes in protein expression in both groups. By using the LC-MS/ MS analysis combined with TMT labelling technology, we identified a total of 3896 proteins that were common to the young and old WKY rat hearts. Proteins with P < 0:05 and FC ≥ 1:2 or FC ≤ 0:83 were considered differentially expressed. In addition, using phos-select iron affinity gel and motif analysis, we identified a total of 1475 phosphoproteins which were common to the young and old WKY rat hearts. Phosphoproteins with P < 0:05 and FC ≥ 1:5 or FC ≤ 0:67 were considered differentially expressed. As shown in Figure 5, ageing induced significant changes in proteins and phosphoproteins. As shown in the heat map in Figures 5(a) and 5(e), compared with young rats, the expression profile in old rats induced 58 proteins and 89 phosphoproteins in proteomics and phosphoproteomics array. Gene ontology analysis ( Figure 5(b)) in proteomics was performed to reveal the strongly enriched biological processes: ion translation process, cellular protein modification process, and protein kinase C-activating G-protein coupled receptor signalling pathway. In addition, we also revealed the strongly enriched molecular function of differential proteins: protein

10
Oxidative Medicine and Cellular Longevity tyrosine/serine/threonine activity, phosphoprotein phosphatase activity, ferroxidase activity, and NAD(P) + proteinarginine ADP-ribosyl transferase activity. Further KEGG analysis ( Figure 5(c)) indicates that these differential pro-teins are also highly enriched in some pathways associated with ageing: adrenergic signalling in cardiomyocytes, apoptosis, necroptosis, protein digestion and absorption, aldosterone synthesis and secretion, insulin secretion,

12
Oxidative Medicine and Cellular Longevity ferroptosis, and viral myocarditis. Gene ontology analysis ( Figure 5(f)) in phosphoproteomics was performed to reveal the strongly enriched biological processes: cellular biosynthetic process, translational initiation, cellular macromolecule biosynthetic process, cellular nitrogen compound biosynthetic process, organic substance biosynthetic process, DNA replication, actin filament organization, regulation of G-protein coupled receptor protein signalling pathway, regulation of cell migration, calcium-dependent cell-cell adhesion via plasma membrane cell adhesion molecules, and signal transduction by the p53 class mediator. Further KEGG analysis ( Figure 5(g)) indicates that these differential proteins are also highly enriched in some pathways associated with ageing: proteoglycans, MAPK signalling pathway, viral carcinogenesis, chemokine signalling pathway, and ErbB signalling pathway. Comparing proteomics and phosphoproteomics data, we found that CaMKII expression increased in both proteomics and phosphoproteomics (Figures 5(d) and 5(h)).

Discussion
Ageing is one of the important risk factors for CVD and is associated with an increased prevalence of ventricular tachyarrhythmia, which contributed to higher morbidity and mortality in the elderly [1]. The prevalence and direct medical costs for CVD were significantly increased in over 65 years of age (especially over 80 years of age) in the United States [2]. Therefore, it is important to explore the mechanisms underlying the ageing process to find approaches to improve it. In this study, we revealed that cardiac remodelling and mitochondrial oxidative stress may contribute to ventricular tachyarrhythmia in the elderly by inducing abnormal Ca 2+ and V m coupling via the NOX4/ROS/CaMKII pathway. It is well known that ageing induces cardiac remodelling even in the absence of underlying pathologies [23,24]. Consistent with published results, we found that ageing exacerbates cardiac fibrosis and impaired cardiac functions in rats. Moreover, our ECG and PES experiments showed significantly increased susceptibility of aged hearts to rapid pacing-induced ventricular arrhythmias, which were in agreement with previous reports [25,26]. In ventricular arrhythmias, fibrosis has been demonstrated to play important roles in creating an essential substrate for these arrhythmias persisting [27]. The published and our present data indicate that ageing-induced fibrosis may be involved in ventricular tachyarrhythmia.
The impairment of electrical activity and conduction has been identified to contribute to cardiac arrhythmia [6, 21, 13 Oxidative Medicine and Cellular Longevity 22]. Using optical mapping assay, we found that the elderly hearts exhibited increased AP and Ca 2+ duration and shortened V m /Ca 2+ latency. Although there have been few studies to investigate the effects of ageing on V m /Ca 2+ latency, growing evidence has identified abnormal myocardial AP and Ca 2+ signal induced by ageing [26,28], which are partly consistent with our results. Previous reports have shown that ATP-sensitive K + channels, delayed rectifier K + channels, and other potassium channels were dysfunctional in the ageing heart, which may contribute to the prolongation of APD and increased the susceptibility to arrhythmias [25,29]. Furthermore, Lei and Huang recently suggested that normal cardiac excitation requires the cyclic events in the intracellular Ca 2+ (Ca 2+ clock) and membrane voltage (membrane clock) homeostasis to be aligned and disruption in this alignment leads to arrhythmia [15]. Our results and the published reports indicate that the shortened V m /Ca 2+ latency and abnormal coupling of Ca 2+ and V m may be involved in the increased ventricular arrhythmias in ageing.
Mitochondria play important roles in cell processes and age-related pathological alterations of the heart. Ageingrelated dysfunction of mitochondria contributed to the development of all common age-related diseases including fibrosis and arrhythmias. We found that the ROS level was increased in the elderly heart, which was also consistent with the previous study [30]. NADPH oxidases (NOXs) were the important source of ROS in the heart, which included NOX1 to 6 subtypes, and NOX2 and NOX4 were the important NOXs in the heart [31]. NOX2 was more expressed in the cell membrane, while NOX4 was predominantly expressed in mitochondria [31]. In our study, it is interesting that NOX4, not NOX2, was increased in the elderly heart (Figures 6(c)-6(e)), which suggested that NOX4 was the main source of increased ROS in the ageing heart. Moreover, it has been shown that ageing significantly inhibited the activity of enzymes that can scavenge ROS, such as SOD [32], indicating ageing-induced ROS generation may also be relative to SOD.
CaMKII, which plays a crucial role in cardiac normal function, can also be oxidized and activated by increased levels of ROS in various cardiac diseases [12,33,34]. There are lots of researches focusing on the activation of CaMKII through phosphorylation. In this study, our phosphoproteomics arrays and Western blotting experiments showed that not only the phosphorylated CaMKII but also the oxidative CaMKII were significantly increased in the elderly hearts, indicating that increased NOX4/ROS may also contribute to the activation of CaMKII through oxidation. Furthermore, CaMKII activation accelerated the Ca 2+ release through phosphorylation of RYR2, which prolonged diastolic relaxation and may induce arrhythmias through DAD via activation of the Na + −Ca 2+ exchanger. Taken together, NOX4/ROS-activated CaMKII may contribute to increased ventricular arrhythmias by inducing abnormal Ca 2+ handling in elderly hearts. In addition, previous studies suggested that the activation of CaMKII was involved in cardiac fibrosis [35,36]. Therefore, activated CaMKII may contribute to ageing-induced cardiac fibrosis and structural remodelling.
NTotally, as shown in Figure 7, in this study, we revealed that cardiac remodelling and activation of the NOX4/ROS/ CaMKII pathway contributed to ventricular arrhythmias in the elderly. More importantly, abnormal Ca 2+ and V m coupling were the important mechanisms involved in the susceptibility to ventricular arrhythmias in ageing.

Data Availability
The data used to support the findings of this study may be released upon application to the corresponding author, Tangting Chen, who can be contacted through the following email: ctt@swmu.edu.cn